Developmental stages and molecular phylogeny of Hepatozoon fitzsimonsi (Dias 1953) (Adeleorina: Hepatozoidae) in tortoises Stigmochelys pardalis (Cryptodira: Testudinidae) and ticks of the genus Amblyomma (Acari: Ixodidae) from South Africa

Background Hepatozoon fitzsimonsi (Dias, 1953) is a frequently found haemogregarine of southern African tortoises. At the time of this species’ reassignment from the genus Haemogregarina to Hepatozoon, developmental stages such as sporocysts and sporozoites were observed in ticks associated with H. fitzsimonsi parasitised and non-parasitised tortoises. It was thus suggested that ticks may act as the potential vectors for this parasite. However, this earlier research was unable to confirm the identity of these sporogonic stages using molecular markers. In a separate study aimed at identifying tick species parasitising South African reptiles and molecularly screening these for the presence of Hepatozoon, that study identified H. fitzsimonsi in tortoise-associated ticks. Thus, the present study aimed to revisit the potential of ticks to act as vectors for H. fitzsimonsi in tortoises using a combined microscopy and molecular approach. Methods Specimens of Kinixys natalensis, Kinixys spekii, Kinixys zombensis and Stigmochelys pardalis were collected from Bonamanzi and Ndumo Game Reserve, South Africa. Upon capture, animals were examined for ticks, and these were collected along with blood and other tissues. Adult ticks were dissected and visceral impression slides were prepared along with thin blood and tissue smears on clean microscope slides. Smears and impression slides were stained with Giemsa, screened and micrographs of parasites were captured. Two primer sets were employed to target fragments of the 18S rRNA gene of parasites found in both tortoises and ticks and the resulting sequences were then compared with other known H. fitzsimonsi and haemogregarine sequences from the GenBank database. Results Peripheral blood gamont and liver merogonic stages were observed in S. pardalis, while the sporogonic stages were observed in the haemocoel of Amblyomma ticks. Gamont and sporocyst stages compared morphologically with previous descriptions of H. fitzsimonsi, identifying them as this species. Phylogenetic analysis revealed that the blood and tick sequences obtained in this study clustered in a monophyletic clade comprising known H. fitzsimonsi. Conclusions The present study provides further support for ticks acting as the vectors of H. fitzsimonsi by molecularly identifying and linking observed developmental stages in tortoises (S. pardalis) with those in the invertebrate host (Amblyomma spp.). Graphical Abstract


Background
Haemogregarines (Apicomplexa: Adeleorina) are commonly encountered protozoan parasites of erythrocytes or leukocytes and are frequently described from a wide variety of vertebrates including amphibians and reptiles [1][2][3][4][5].Even though this group of parasites contains several genera, such as Hemolivia Petit et al. 1990, Haemogregarina Danilewsky 1985 and Karyolysus Labbé 1894, the genus Hepatozoon Miller 1908 appears to be the most prevalent in reptiles [3,6].However, even with the increase in research on these parasites in the last decade, knowledge on the diversity and systematics of these apicomplexans is still lacking and remains contentious [4,7].Based on relationships of these haemogregarine genera inferred using the 18S ribsomal RNA (rRNA) gene, Hepatozoon remains paraphyletic [5][6][7][8][9][10][11].This lead Karadjian et al. [12] to erect a new genus Bartazoon Karadjian et al., 2015, to resolve this issue.Species of Hepatozoon display a heteroxenous life cycle, requiring a vertebrate host in which asexual reproduction occurs, as well as an invertebrate host in which sexual reproduction occurs [6,12,13].Invertebrate hosts range from haematophagous insects to acarine hosts such as ticks and mites [12].For transmission to occur between the parasitised vector and the vertebrate host, ingestion of this vector by the vertebrate is required or ingestion of tissue cysts in a vertebrate host is required [1,12].Karadjian et al. [12] proposed that Bartazoon would comprise species vectored by haematophagous insects, whilst those species vectored by acarine hosts would remain as Hepatozoon.However, Bartazoon has up to now not been widely accepted as the monophyly of this genus is not well supported [7,11,14].
Hepatozoon fitzsimonsi (Dias, 1953), a haemogregarine of southern African tortoises, along with other Hepatozoon species of amphibians and reptiles, was one of the species proposed to be a member of Bartazoon [12].This, as such, suggested the potential vector of this parasite to be a biting insect.However, research done on this parasite persistently observed a close association with tortoises and ticks [3,15], the preceding study observing what appeared to be sporogonic stages including sporocysts and sporozoites in tick haemocoel from infected and uninfected tortoises.Even though Cook et al. [3] attempted to molecularly identify these tick stages, it was unsuccessful.Recently, two molecular screening studies [11,16], both screening ticks collected from reptiles in Kenya and South Africa for tick-borne pathogens and Hepatozoon, respectively, identified H. fitzsimonsi in ticks.In both studies unengorged ticks were used.With these previous findings in mind, the present study aimed to revisit the potential that ticks can act as vectors for H. fitzsimonsi in tortoises, by (i) collecting blood/tissue and ticks from tortoises, (ii) screening both microscopically for the presence of blood, merogonic and sporogonic stages, respectively, and (iii) molecularly characterising these stages to determine if they are that of H. fitzsimonsi.The present study thus provides further support for ticks acting as the vectors of H. fitzsimonsi based on observation of its life cycle stages in tortoises (Stigmochelys pardalis) as well as in the invertebrate host (Amblyomma spp.).

Tortoise and tortoise tissue collection, and preparation of blood and other tissue slides
Tortoises were collected during 2016-2017 from Bonamanzi and Ndumo Game Reserve, KwaZulu-Natal (KZN), and identified to species level using field guides [17][18][19].As these animals are protected by law, permits did not allow for euthanasia; however, in one case a tortoise was found recently dead, the result of a roadkill incident.
Blood was collected from the subcarapacial sinuses [20] and thin blood smears were prepared, and allowed to air dry before being fixed in absolute methanol and stained in a solution of Giemsa stain (FLUKA, Sigma-Aldrich, Steinheim, Germany) for 20 min [3].Once stained and dried, these were stored in a dustproof container for later screening.A small volume of blood was preserved in 70% ethanol for molecular work.Samples from the roadkill individual were taken from the kidney, heart, liver, lung, and spleen during dissection.Impression and squash smears were prepared on clean microscope slides, and these fixed and stained following the same method as described for the blood smears.A small section of each organ was preserved in 70% ethanol.

Tick collection, with preparation of tick impression slides
Ticks present on tortoises at the time of blood collection were carefully removed using forceps to ensure that the hypostome remained intact with minimal damage to the tick.Ticks were placed in plastic tubes and labelled according to the individual tortoise from which they were collected.Ticks were allowed to digest their blood meals for approximately 10-20 days [13,21].Thereafter, adult ticks were dissected according to Edwards et al. [22], and visceral impression slides were prepared on clean microscope slides.The remaining tick and its viscera were immediately fixed in 70% ethanol and stored in a −20 °C freezer until further molecular analysis.Slides were allowed to dry and followed the same fixing and staining protocol as that of the blood and organ smears.Morphological identification to species level was done with the aid of a Nikon AZ100M microscope and guidelines for tick identification as provided by Theiler [23] and Theiler and Salisbury [24].

Microscopical screening of tortoise blood and tissue smears and tick impression slides
Stained blood smears were screened at 100 × oil immersion objective, and micrographs of parasites were taken on a calibrated Nikon Eclipse E800 compound microscope (Nikon, Amsterdam, the Netherlands) using the imaging software NIS Elements.All measurements were in micrometres (µm) unless otherwise indicated.Measurements comprised the parasite's length (including recurved tail when present) and width within its parasitophorous vacuole (PV), and the parasite's nucleus length and width [3].Subsequently, the morphometric data of parasites were used in comparison to previous descriptions of H. fitzsimonsi.
Impression slides were screened using the 20×, 60× and 100× oil immersion objective on a calibrated Nikon Eclipse E800 compound microscope (Nikon, Amsterdam, the Netherlands) using the imaging software NIS Elements.Micrographs of sporogonic stages were taken using both the 20× and 100× objectives, depending on the size of the parasite stage.Sporogonic stages were identified and measured according to Cook et al. [3], with the aid of the ImageJ version 1.47 software program (Wayne Rasband National Industries of Health, USA) [25] (http:// imagej.nih.gov/ ij) and subsequently compared with those observed in ticks from Cook et al. [3].The number of sporozoites within each sporocyst were estimated by counting their nuclei [3].

DNA extraction, PCR and phylogenetic analysis
Genomic DNA was extracted from the blood and liver samples of tortoises, as well as from visceral samples of ticks using the KAPA Express Extract Kit (Kapa Biosystems, Sigma-Aldrich).Once extracted, DNA was used for polymerase chain reaction (PCR) amplification, amplifying approximately the full 18S rRNA gene in two fragments for the tortoise samples by using a combination of primer sets.The first fragment, approximately 930 nt in length, was amplified using primer set HAMF 5′-GCC AGT AGT CAT ATG CTT GTC-3′ [26] and HepR900 5′-CAA ATC TAA GAA TTT CAC CTC TGA C-3′ [27].The second fragment, approximately 1400 nt in length, was amplified using primer set HepF300 5′-GTT TCT GAC CTA TCA GCT TTC GAC G-3′ [27] and 2868 5′-TGA TCC TTC TGC AGG TTC AC-3′ [28,29].
Conditions for PCR of both fragments were as follows: initial denaturation at 95 °C for 3 min, followed by 35 cycles, entailing a 95 °C denaturation for 30 s, annealing at 61 °C for 30 s with an end extension at 72 °C for 2 min, and following the cycles a final extension of 72 °C for 10 min [30].
Difficulties were encountered using the above primer sets and protocols for ticks (only that for Amblyomma marmoreum was partially successful), as such for the tick visceral samples, fragments of the 18S rRNA gene were amplified using the primer set HepF300 5′-GTT TCT GAC CTA TCA GCT TTC GAC G-3′ and HepR900 5′-CAA ATC TAA GAA TTT CAC CTC TGA C-3′, targeting a fragment of approximately 600 nt.
Conditions for PCR were as follows: initial denaturation at 95 °C for 3 min, followed by 35 cycles, entailing a 95 °C denaturation for 30 s, annealing at 60 °C for 30 s with an end extension at 72 °C for 1 min, and following the cycles a final extension of 72 °C for 10 min as detailed according to previous methods [31].
PCR reactions were performed with volumes of 25 μL, using 12.5 μL Thermo Scientific DreamTaq PCR master mix (2×) (final concentration: 2× DreamTaq buffer, 0.4 mM of each dNTP and 4 mM MgCl2), 1.25 μL (10 μM) of each of the primer sets mentioned above, and at least 25 ng DNA.The final reaction volume was made up with PCR-grade nuclease-free water (Thermo Scientific).Reactions were undertaken in a Bio-Rad C1000 Touch ™ Thermal Cycler PCR machine (Bio-Rad, Hemel Hempstead, UK).Resulting amplicons were visualized under ultraviolet light on a 1% agarose gel stained with gel red using a Bio-Rad GelDoc ™ XR + imaging system (Bio-Rad, Hemel Hempstead, UK).PCR products from each sample were sent to a commercial sequencing company (Inqaba Biotechnical Industries (Pty) Ltd, Pretoria, South Africa) for purification and sequencing in both directions.Quality of resultant sequences was assessed using Geneious Prime ® 2022.2.2 [32] (http:// www.genei ous.com) before consensus sequences were generated from both forward and reverse sequence reads for both fragments.A consensus sequence was then generated from both fragments.Sequences were identified using the Basic Local Alignment Search Tool (BLAST) (http:// blast.ncbi.nlm.nih.gov/).
Phylogenetic analysis involved comparative sequences of species of Dactylosoma, Karyolysus, Haemogregarina, Hemolivia and Hepatozoon with Adelina dimidiata, Adelina grylli and Klossia helcina (GenBank: DQ096835, DQ096836, MT094865) as outgroup were downloaded from GenBank and aligned to the sequences generated within this study.Sequences were aligned using the MUSCLE alignment tool [33] implemented in Geneious Prime ® 2022.2.2.The alignment consisted of 60 sequences and was 1261 nt long.To infer phylogenetic relationships of the aligned dataset the Bayesian Inference (BI) method was used.
A model test was performed to determine the most suitable nucleotide substitution model using the Smart model selection software [34] (https:// www.atgc-montp ellier.fr/phyml-sms/).The best model identified was the General Time Reversible model with estimates of invariable sites and a discrete Gamma distribution (GTR + I + Γ).The BI analysis was implemented from within Geneious Prime ® 2022.2.2 using MrBayes 3.2.2[35].The analysis was run twice over 10 million generations for the Markov Chain Monte Carlo (MCMC) algorithm, with a subsampling frequency of 200 generations and a 'burn-in' of 25%.Pairwise distances were generated by Mega-X 10.2.4 with a Kimura two-parameter substitution model with transitions and transversions included [36,37], as in Zechmeisterová et al. [38].

Results
A total of 14 tortoises were collected, one Kinixys natalensis and one Kinixys spekii, seven Kinixys zombensis and five Stigmochelys pardalis.A total of 10 of the 14 (71%) tortoises were infested with ticks including larval, nymphal and adult stages.Larval and nymphal squashes yielded no identifiable haemogregarine stages and as such adult ticks formed the focus of this study.As species of Kinixys are often infected with both H. fitzsimonsi and Hemolivia parvula, three of the S. pardalis were selected for further analysis.Of these, one showed no evidence of haemogregarine peripheral blood stages [S.pardalis (1)], the second [S.pardalis (2)] showed an extremely low parasitaemia infection (0.1%) and the third [S.pardalis (3)] showed a relatively high parasitaemia (5%), of what was morphologically comparable with gamonts of H. fitzsimonsi (Fig. 1A).Within the third tortoise, the roadkill individual, merogonic stages were observed in the liver (Table 1).No observable stages were identified in the other organ smears.
Three species of Amblyomma ticks (Acari; Ixodidae) were collected from the three S. pardalis; these were Amblyomma herbraeum, Amblyomma marmoreum and Amblyomma nuttalli.Impression slides from all three species showed sporogonic stages within the haemocoel.A single intact sporulated oocyst was observed in A. marmoreum (Table 1).No gametogenesis and subsequent fertilisation were observed in any of the ticks.
Remarks Gamont stages in the present study were morphologically comparable with those described by Dias [39]   (17-17.5 × 3.4-4.2).These were the only peripheral blood stages found in the present study.Meront stages were observed only in the liver, unlike that of other species of Hepatozoon, which may be observed also infecting various other internal organs such as the lungs [6,13,40,41].In the present study, the number of merozoites contained within meronts could not be conclusively determined, presumably due to the thick non-staining capsule.However, as numbers were low, this may suggest that they represent macromeronts [6].Even so, the number of merozoites contained in macromeronts is highly variable among species of Hepatozoon.For instance, in species Hepatozoon kisrae Paperna, Kremer-Mecabell and Finkelman 2002 of the agamid Laudakia stellio, macromeronts divided into four to 16 macromerozoites [40], whilst in the species Hepatozoon domerguei Landau, Chabaud, Michel, and Brygoo 1970 of snakes, mature macromeronts could contain approximately 30 macromerozoites [6].No cyst or cyst-like stages were observed in the present study, these stages often observed in the liver and lung tissue of snakes and lizards infected with Hepatozoon, such as in the case of H. domerguei [6].

Phylogenetic analysis of blood and tick stages
A 1733 nt and 1688 nt sequence was achieved for the Hepatozoon infecting S. pardalis (2) and S. pardalis (3) (GenBank accession numbers: PP718267, PP718268 respectively).A 626 nt sequence was isolated from A. nuttalli collected from S. pardalis (1), along with a 620 and 626 nt sequences for A. herbraeum collected from S. pardalis (2) (GenBank accession numbers: PP718266, PP718263, PP718264 respectively).From A. marmoreum, collected from S. pardalis (3), a 994 nt sequence was isolated (GenBank accession number: PP718265).Between all sequences isolated from these ticks and the blood of tortoises, genetic divergences were 0%, strongly suggesting that the blood stages and those in the ticks represent the same haemogregarine species.
Comparisons of those isolates from the current blood stage material [S.pardalis (2) and (3)] with previous molecular descriptions of H. fitzsimonsi (KJ702453, KR069084), showed a genetic divergence of 0-0.2% (Table 2).This was the same for the isolates sequenced from the ticks in the current study; the blood and tick isolates clustered in a monophyletic clade consisting of the known H. fitzsimonsi (Fig. 3).Genetic divergence was 0% for that collected from both tortoise blood and ticks with A. sparsum collected off tortoises in Kenya, clustering within the same H. fitzsimonsi clade.Hepatozoon cuorae (Chai and Chen, 1990) [38,43] showed the closest relationship with the isolates from this study, with a divergence of 0.2-0.6%(Table 2).The Hepatozoon sp.sequence from Kinosternon scorpioides showed variable divergence (0.2-1.1%) between sequences of this study and known H. fitzsimonsi (Table 2), which was dependent on length of sequence and coverage.Both H. cuorae and the latter Hepatozoon sp.clustered as sister taxa to the H. fitzsimonsi clade.Divergence between Hepatozoon simidi Gutiérrez-Liberato, Lotta-Arévalo, Rodríguez-Almonacid, Vargas-Ramírez, and Matta 2021 [44] and the current material was 3%, clustering separately and basal to a clade containing Hepatozoon of squamata (Fig. 3).

Remarks
As the isolates of the present study cluster primarily with known H. fitzsimonsi and within a larger clade containing Hepatozoon of other chelonians, those isolates from unengorged ticks containing sporogonic stages are considered representative of H. fitzsimonsi.Where a divergence of 0.2% was noted between sequences of H. fitzsimonsi, this would be because of comparisons between shorter and longer sequences.Sequences from ticks were shorter than desired as the primer set HepF300 and HepR900 was used instead of the primer sets and protocol used for the tortoise blood (which produced longer sequences).However, this primer set does amplify a fragment of a highly variable region in the 18SrRNA gene and is therefore adequate for phylogenetic analysis [38].Alignment of the present study's sequences and those of known H. fitzsimonsi were identical but for one base pair in one sequence, when comparing this region, confirming the haemogregarine within the ticks to be H. fitzsimonsi.The low support for this clade is therefore presumed to result from low evolutionary signal or variation among the sequences.Comparisons of H. fitzsimonsi from tortoise blood to H. cuorae showed a divergence of 0.5-0.6% for comparatively long sequences (H.cuorae: 1715 nt; H. fitzsimonsi tortoise 2 and 3: 1733 nt and 1688 nt, respectively), confirming that they are separate species whilst taking into consideration the conservative nature of the 18S rRNA gene.Regarding the placement of H. simidi with Hepatozoon spp. of squamata, this too was observed by Zechmeisterová et al. [38], where this species was observed in these authors' phylogenetic analysis with a Hepatoon sp. from a snake.

Discussion
Hepatozoon fitzsimonsi was the first chelonian haemogregarine to be reassigned to the genus Hepatozoon on the basis of morphological and molecular findings [3].In the years that followed, two other species of Hepatozoon were described and reassigned from chelonians, Hepatozoon simidi and Hepatozoon cuorae, respectively [38,44].Further reports for Hepatozoon in chelonians include an unpublished speculative sequence of H. fitzsimonsi from an unnamed Kinixys sp. from Nigeria, a sequence of H. fitzsimonsi from the tick Amblyomma sparsum from tortoises from Kenya  [11,16,38], and the Hepatozoon sp. reported from Gopherus polyphemus [45] and Sternotherus odoratus [46] in the USA.As mentioned by Zechmeisterová et al. [38], two sequences, named as Hepatozoon on Gen-Bank, from Mauremys leprosa by Marzal et al. [47], are in fact representatives of Haemogregarina.For the three morphologically described and molecularly supported species of chelonian Hepatozoon above, the only species for which a potential definitive host has been provided is H. fitzsimonsi [3,38,44].The present study found sporogonic stages in three tick species of the genus Amblyomma, which were morphologically comparable with those found in ticks by Cook et al. [3], linking these molecularly to H. fitzsimonsi.Sporocyst stages in the present study were on average longer and much wider than those described in Cook et al. [3]; this was particularly evident when observing the range in length and width of sporocysts.Simultaneously, 61 sporocysts were measured in the present study as compared with the 10 in Cook et al. [3].Furthermore, sporocysts were observed in the present study in what appeared to be various stages of development: were mature and contained developed sporozoites, others had a residual body still present, and others were smaller and more spherical.At times sporocysts were seen which appeared to have no sporozoite development within and, equally, sporozoites were seen that appeared to be degrading or observed just prior to division, the former potentially representing an older phase of infection.The variability in size and shape of sporocysts, besides being a potential result of development phase, appears to be typical in Hepatozoon between species [13] and is also evident within species -Hepatozoon rarefaciens (Sambon and Seligmann, 1907) [48]    Hepatozoon fitzsimonsi ex Amblyomma herbraeum (2) 0.028 0.028 0.030 0.002 0.002 0.002 0.000 0.000 0.000 0.000 0.000 0.000 14 Hepatozoon fitzsimonsi ex Amblyomma marmoreum ex Stigmochelys pardalis (3) 0.025 0.022 0.030 0.009 0.002 0.002 0.000 0.000 0.000 0.000 0.000 0.000 0.000 Votýtpka and Modrý 2007 [41] (20-53 × 18-42) [6].Two purple-stained structures were seen on either side of the nucleus of sporozoites in the material of Cook et al. [3], which were not seen in the present study's sporozoites; however, this may be as a result of staining bias.Regardless, as sequencing of tick stages in Cook et al. [3] was unsuccessful and thus it cannot be ascertained that the infection seen in this previous study was representative of H. fitzsimonsi.Only a single intact oocyst was observed in the material of the present study, regardless of the care taken to be as gentle as possible when preparing the impression slides.This, and the thin oocyst wall, suggests that these stages rupture readily, as in Hepatozoon clamatae Stebbins [51] of frogs.Similarly, as with sporocysts, oocysts too show a high variability in dimensions between species [13]; however, as only one oocyst was found, it cannot be determined if this may also be at an intra-species level.
Meronts observed in the liver of the one tortoise were tentatively identified as macromeronts.No larger meronts containing higher numbers of merozoites, which may have represented micromeronts, were observed in the current material.Macromeronts are considered the first stage of merogonic development in species of Hepatozoon, which eventually give rise to micromeronts, the micromerozoites of which will in time enter erythrocytes to form gamonts [13].As only mature gamonts were observed in the peripheral blood of the tortoise, with no younger stages such as trophozoites, along with the lack of probable micromeronts in the liver, it may be suggested that these indicate two separate infections.Both macro-and micromeronts have not been observed for any species of Hemolivia so far [52], including Hemolivia mauritanica, a species infecting terrestrial tortoises of the western palearctic realm [52,53].Hemolivia parvula (Dias, 1953) has been found to infect terrestrial tortoises of eastern southern Africa.However, naturally it appears to infect only species of Kinixys, with only a single finding in a captive S. pardalis [3,15,54].Regardless, as the risk of co-infection, particularly in chelonian hosts, is high, as is evident in the study by Zechmeisterová et al. [38], care needed to be taken that stages seen in both tortoise and tick hosts were not that of Hm. parvula.As such, during the analyses of the raw sequence data, chromatograms were checked for evidence of double peaks, which would be suggestive of co-infections.As no double peaks were observed and the quality of sequences was high, it supports that the merogonic stage seen in the liver of the present material is most likely that of H. fitzsimonsi and not Hm.parvula.Compared with other species of Hepatozoon and species of Hemolivia, in which cystic stages are often present [6,13,52], no cystic stages were observed in the current study.It has been suggested that these stages aid in the persistence of infection [6,52] and may play a role in transmission of infection from one vertebrate to another, particularly in three host life cycles [6].However, these scenarios require further investigation at a more individual level and cannot be considered a general rule.
Karadjian et al. [12] suggested that H. fitzsimonsi be reassigned as a species of the then newly erected genus Bartazoon, which is transmitted by biting insects.Even though infection of more than one group of arthropod hosts cannot be ruled out at this stage, particularly given the findings on Hepatozoon fusifex Ball et al. [55], which showed infection in its natural tick host along with experimentally infected mosquitoes of widely different origin [6], it appears rare.For instance, in the dissection of 100 ticks feeding on H. ayorgbor-infected snakes, only the mosquitoes were found to contain sporogonic stages [6,41].Equally, attempts by Morsy et al. [56] to detect sporogony of the snake haemogregarine species Hepatozoon seurati (Laveran and Pettit 1911) in mites, ticks, sand flies and Aedes aegypti fed on infected snakes, were unsuccessful with stages found only in epidemic Culex pipiens molestus [6,56].This was equally true for Hepatozoon tupinambis (Laveran and Salimbeni 1909), where attempts to infect leeches and triatomid bugs on infected teiid lizards was unsuccessful [6].Given that sporogonic stages, with the final infective developmental stage, the sporozoite, has been identified in unengorged Amblyomma ticks and linked molecularly to H. fitzsimonsi in this study, it is assumed that these or at least three species of this genera can act as definitive hosts and vectors for H. fitzsimonsi.However, the transmission capability of these ticks will need further investigation, as well as to how tortoises become infected.Tortoises do display aggressive behaviour during courtship or when competing for a mate, often biting the other tortoise.This can be observed in Desert tortoises in the USA, as well as in tortoise species in South Africa [57], Cook, personal observation].It may be in such cases where ingestion of infected ticks occurs.If species of Amblyomma are vectors for H. fitzsimonsi, which these findings highly suggest that they are, H. fitzsimonsi does not fit within the taxonomic description of Bartazoon (transmitted only by biting insects), making the genus paraphyletic.

Conclusions
As mentioned previously, H. fitzsimonsi was the first haemogregarine of chelonians to be reassigned from Haemogregarina to Hepatozoon on the basis of on morphological and molecular findings.This was particularly significant as it broadened the range of potential vectors for this species and was therefore no longer limited to leeches.Even though Cook et al. [3] did find sporogonic stages in ticks associated with tortoises infected with H. fitzsimonsi, these parasites could not be confirmed as definitive hosts.In the present study sporogonic stages in three species of Amblyomma ticks were molecularly confirmed to be that of H. fitzsimonsi, substantiating that these parasites do act as definitive hosts and strongly suggesting that they are a vector.Even though further research into the transmission of H. fitzsimonsi from ticks to tortoises may be required, this would be the second haemogregarine of tortoises for which ticks are the vector, with Hemolivia mauritanica being the first.Importantly, this is the first species of Hepatozoon infecting chelonians for which ticks have been identified as a definitive host.This will hopefully encourage further research, identifying definitive hosts and vectors for other species of chelonian Hepatozoon, a research area that remains largely neglected at present.

Fig. 2
Fig. 2 Sporogonic stages in the haemocoel of Amblyomma nuttalli and Amblyomma herbraeum collected from Stigmochelys pardalis (1) and (2), respectively.A Sporocyts in A. nuttalli, one showing developed sporozoites, the other showing a central residual body (arrow).B-D Sporocysts and sporozoite in A. herbraem, B showing larger and smaller more ovoid sporocyst (arrowhead), and either sporocysts prior to division or in the process of degrading (arrow), and C showing a mature sporocyst with developed sporozoites (arrowhead), a developing/maturing and undeveloped sporocyst.D Sporozoite (circled), surrounded by a clear membrane, showing a nucleus (arrow) and potential crystalline body (arrowhead).Scale bar, 10 µm

Fig. 3
Fig. 3 Phylogenetic tree of haemogregarine partial 18S rDNA sequences (60 sequences and 1261 nt) based on Bayesian inference (BI) analysis.Nodal supports shown as Bayesian posterior probabilities.Blue clade showing species of Hepatozoon of chelonians, including Hepatozoon fitzsimonsi from this (bolded) and previous studies.Green sequence is that of Hepatozoon simidi isolated from a terrapin species from Brazil.It is the only Hepatozoon not included in the main chelonian Hepatozoon clade.Outgroup comprised Adelina dimidiata, Adelina grylli and Klossia helcina (GenBank: DQ096835, DQ096836, MT094865).The scale bar represents 0.04 nucleotide substitutions per site

Table 1
Summary of haemogregarine infection, tick species collected and screened for sporogonic stages, in and from three individual tortoises selected for analysis −, negative; +, positive; n, number

Table 2
Genetic distances between species of Hepatozoon infecting chelonians with species of Hemolivia as reference for genetic distance between genera.Sequences 9-14 are those of the current study